Project Summary: Project 1 aims to use polyclonal recipient Tregs in a compressed conditioning protocol to achieve mixed chimerism and tolerance to deceased-donor kidneys and/or islets. Islet transplantation for the cure of Type 1 diabetes (T1D) is currently limited by low 5-year survival rates. Poor islet survival is likely the result of islet loss due to alloimmune responses, recurrent autoimmunity, and toxicity from immunosuppressive drugs. Thus, this therapy can only ethically be offered to patients with life-threatening complications of T1D. Inducing tolerance to islets through the development of mixed chimerism has the potential to eliminate both alloimmune and autoimmune islet loss, as well as the need for immunosuppressive medications. There are several barriers to inducing mixed chimerism and tolerance to kidneys and/or islets using deceased donors that we aim to overcome. Our current tolerance induction regimen, developed in cynomolgus monkeys and translated to humans, involves a 6-day conditioning of the immune system prior to organ transplant and is therefore only applicable to living donation. Previous attempts to compress immune conditioning to 24 hours, to allow the use of deceased donors, failed to induce chimerism or tolerance. Additionally, durable chimerism may be required for reversal of autoimmunity in T1D patients and might then be necessary for optimal islet survival post- transplant. Since the kidney has been shown to contribute to tolerance in the case of transient chimerism, it is even more important to achieve durable chimerism in recipients of islet transplants without co-transplantation of a donor kidney if tolerance is to develop. We now have data showing that expanded polyclonal recipient Tregs can achieve markedly prolonged chimerism and more robust tolerance than has previously been possible in the cynomolgus model. We hypothesize that the addition of polyclonal recipient Tregs, that can be prepared in advance, to a compressed conditioning regimen, will permit the development of mixed chimerism and tolerance to kidneys and/or islets. Our approach targets patients with end-stage renal disease alone (Aim 1), patients with T1D and renal failure (Aim 2A), and finally T1D without renal failure (Aim 2B). Therefore, additional measures will be added in Aim 2 if durable chimerism is not achieved in Aim 1. Finally, accurate characterization of the mechanism(s) of tolerance following transient or durable chimerism in nonhuman primates is lacking, in large part due to the absence of precise tools to characterize deletional and regulatory mechanisms. In Aim 3, we will use standard immunologic assays combined with the high-throughput TCR CDR3 tracking system developed in Core B (based on the human TCR platform developed in this laboratory and modified for cynomolgus monkeys) to accurately quantify deletion/expansion of donor-reactive effector T cells and donor-specific Tregs. Islets will be provided by Core A and collaboration with Project 2 and the cores will be coordinated through Core C.